1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of yeast and particularly Dekkera bruxellensis (a.k.a. Brettanomyces) in wine. More specifically, this invention relates to novel probes, probe sets, methods and kits that can be used to detect, identify and/or quantitate (enumerate) one or more yeast in a sample and particularly those organisms that spoil wine.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, identification, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from yeast, eucarya, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result of difficulties associated with probe stability, specificity, sensitivity and reliability.
Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide that can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365: 566–568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes that are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions that strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc. 118:5544–5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay, appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8:53–63, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792–2799 (1997)).
Though they hybridize to nucleic acid with sequence specificity (See: Egholm et al., Nature, at p. 567), PNAs have been slow to achieve commercial success at least partially due to cost, sequence specific properties/problems associated with solubility and self-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S., Tett. Lett. 36:6823–6826 (1995), Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P. E., Angew. Chem. Int. Ed. Engl. 35:1939–1942 (1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and Weiler, K., Nucleosides & Nucleotides 16:1775–1779 (1997) at p 433, col. 1, ln. 28 through col. 2, In. 3) as well as the uncertainty pertaining to non-specific interactions that might occur in complex systems such as a cell (See: Good, L. et al., Antisense & Nucleic Acid Drug Development 7:431–437 (1997)). However, problems associated with solubility and self-aggregation have recently been reduced or eliminated (See: Gildea et al., Tett. Lett. 39: 7255–7258 (1998)). Nevertheless, their unique properties clearly demonstrate that PNA is not the equivalent of a nucleic acid in either structure or function. Consequently, PNA probes need to be evaluated for performance and optimization to thereby confirm whether they can be used to specifically and reliably detect a particular nucleic acid target sequence, particularly when the target sequence exists in a complex sample such as a cell, tissue or organism.
DNA and PNA probes targeting rRNA have been used for the detection of bacteria (gonorrhoeae and mycobacteria) and eucarya by in situ hybridization (See: WO95/32305 (now U.S. Pat. No. 5,985,563), WO98/15648; and WO97/18325 (now U.S. Pat. No. 5,888,733) respectively). PNA probes have also been used to examine telomeres and repeat sequences by in-situ hybridization (See: WO97/14026). Methods for the linking of enzymes to both DNA and PNA probes are known in the art (See: WO99/41273). However, the use of enzyme-labeled DNA probes for the detection of yeast cells by in-situ hybridization has not yet been demonstrated (Amann, R. I., Zarda, B., Stahl, D. A. and Schleifer, K.-H., Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes, Applied and Environmental Microbiology, 58: 3007–3011 (1992)) and Applicants are unaware of any attempts to use enzyme-labeled PNA probes to detect yeast by in-situ hybridization. The lack of examples of successful ISH assays utilizing enzyme linked probes likely results because of difficulties in getting such large molecules to pass through the cell membrane into the yeast cytoplasm.
Wine making is both a hobby as well as an established industry. The organism called Brettanomyces (ascosporic state of Dekkera) within the wine industry is a spoilage yeast causing ‘mousiness’; an undesirable odor and taste. The nomenclature of Brettanomyces used within the field of wine enology is based on synonyms rather than on accepted specie names from the recently published taxonomy of the Brettanomyces/Dekkera species (See: Smith, M. T., The Yeasts A Taxonomic Study (eds. C. P. Kurtzman & J. W. Fell), Elsevier Science B. V., Amsterdam, The Netherlands (1998), pp. 174–177 and pp. 450–453). The lack of proper characterization of the organism that causes wine spoilage has complicated the nucleobase sequence design of specific probes targeting the spoilage organism, since available rDNA sequence information (See: Kurtzman, C. P. and C. J. Robnett., Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences, Antonie van Leeuwenhoek 73: 331–371 (1998), Boekhout, T., Kurtzman, C. P., O'Donell, K. and Smith, M. T., Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences, International Journal of Systematic Bacteriology, 44: 781–786 (1994)) is based on the five accepted species of Brettanomyces/Dekkera (Dekkera anomala, Dekkera bruxellensis, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus). Therefore, it is not obvious what sequence information should be used to generate target sequences suitable for specific detection of the wine spoilage organism known in the wine industry as Brettanomyces. 
Current methods for identification and enumeration of Brettanomyces in wine takes 1–2 weeks and rely on growth occurring on a semi-selective culture medium followed by final identification from morphology and biochemical testing (See: Fugelsang, K. C., Wine Microbiology Chapman & Hall, NY (1997)). This process is very time consuming and relies on highly trained laboratory personal to perform the final identification. Faster methods are available for enumeration of organisms present in a sample but these methods lack the ability to identify or speciate the detected organisms. Because wine samples often contain non-spoilage yeasts, such as Saccharomyces cerevisiae, these methods are of limited usefulness. Because wine spoilage caused by Brettanomyces is a substantial concern for those who make wine, any methods, kits or compositions suitable for rapid, reliable and sensitive detection, identification and/or quantitation (enumerate) of Brettanomyces in wine would allow for more effective intervention in the wine making process and thereby improve product quality and/or reduce the costs of manufacture.